The antigen-antibody reaction is the basis for all immunological test methods. Special proteins called antibodies are produced by an animal in response to the presence of an antigen, that is a foreign protein, in the body fluids of the animal. This normal body response to a foreign protein has led to the development of a number of techniques which are used to diagnose various human and animal diseases or disorders. Immunological test methods may also be used to detect pregnancy. In vitro tests for the presence of a suspected antigen or antibody in a body fluid are carried out by adding the immunological counterpart to a vial of the body fluid, i.e. add antigen if the test is for the presence of antibody or add antibody if the test is for the presence of antigen. If the suspected protein is present the resulting antigen-antibody reaction is generally indicated by precipitation or agglutination of the antigen-antibody complex. As used herein the term body fluid refers to urine, serum, plasma, or the like.
In some instances the antigen-antibody complex is slow to form and the particles that are formed are too small to be observed with certainty. In such cases, detectability of the antigen-antibody reaction can be improved by utilizing a carrier. When the antigen or antibody is coated on the surface of a carrier the reaction with the immunological counterpart produces a visible mass or agglutant. The proteinic antigen or antibody may be adsorbed onto the surface of carriers such as erythrocytes, bacterial cells, bentonite, polystyrene latex particles, anionic phenolic resins, or finely divided diazotized amino cellulose. It has been found however, that chemical binding of the antigen or antibody molecule to the carrier is superior to physical adsorption.
Immunological test methods for proteins such as human chorionic gonadotropin (HCG) and myoglobin may be useful as an aid in medical diagnosis.
A number of immunological tests have been developed for the detection of HCG in urine or serum. HCG is a glycoprotein with a molecular weight of about 27,000 produced by the chorionic tissue of the placenta during pregnancy, Systems using HCG have been used to detect pregnancy. During pregnancy this hormone governs the production and secretion of progesterone by the corpus luteum. HCG is also produced in large quantities by hydatidiform moles, choriocarcinomas, and some tumors of the tests. Low levels of HCG have also been found by radioimmunoassay in the sera of patients with various nontrophoblastic neoplasms. Various agglutination techniques have been used to test for the presence of HCG.
Agglutination testing for HCG may be performed by either the indirect or the direct technique. In the indirect technique the clinical sample is mixed with HCG antibody at a dilution that will be completely bound by one or more International Units/Milliliter (I.U./ML) of HCG. After an initial incubation period an indicator system consisting of HCG bound to a particulate carrier (latex or red cells) is added to the mixture. If HCG is present in the clinical sample the HCG antibody will not be available to react with the HCG-carrier complex and there will be no agglutination, thus, absence of agglutination is a positive test for HCG. If, on the other hand, HCG is not present in the clinical sample the HCG antibody will react with the HCG-carrier complex causing agglutination of the indicator system. This is a negative test for HCG in the clinical sample. In the direct technique HCG antibody bound to the carrier reacts directly with the HCG in the clinical sample and there is no need for an intermediate incubation step. Thus, in the direct technique agglutination indicates a positive test for HCG in the clinical sample.
Myoglobin is a muscle protein resembling hemoglobin in that myoglobin can reversibly bind oxygen. The concentration of cardiac myoglobin has been found to be increased in serum and urine after myocardial infarction. This increase in myoglobin concentration has been used as a diagnostic indicator of acute myocardial infarction. Increased levels of myoglobin can be detected hours before the elevation of cardiac enzymes thus serving as an early aid to diagnosis. The greatest levels of urinary myoglobin appear within hours of onset of clinical symptoms. Urinary myoglobin is a specific and sensitive diagnostic indicator of serious renal and respiratory failure as well, Hibrawi, H. and R. G. Blaker, Clin. Chem. 21/6:765 (1975), and methods of measurement have included radioimmunoassay, immunofluorescence, radial immunodiffusion, complement fixation, counterimmunoelectrophoresis and hemagglutination tests. All of these methods incorporate the use of an antibody derived from immunizing animals with a human myoglobin preparation.
It would be highly desirable to provide an effective, easy to read diagnostic test for the detection of particular proteins.
The prior art teaches that polyacrylamide can be functionalized by treatment with glutaraldehyde to which ligands bearing amino groups may be bound as Schiff's bases, Ternynck, et al., F.E.B.S. Letters, 23, 24 (1972). The present invention utilizes the hydrazide group which is more nucleophilic than the primary amide group, therefore the reactions of hydrazides, as embodied in this invention, can be carried out with greater ease under milder conditions and with a larger variety of functional groups. When working with a water-based colloid, such as a latex, this greater reactivity is a substantial advantage.
The prior art also teaches that amino-bearing bodies such as peptides and proteins can be attached chemically to polymeric water-insoluble carriers bearing hydrazine residues via dialdehydes, German (West) Patent DT No. 2,530,247 (1976). These water insoluble carriers include agarose polymer, acrylamide, polyethylene/maleic anhydride polymer, cellulose, a substituted cellulose, polyamide and glass. It is physically possible to handle these polymers by routine laboratory manipulations as would be employed with many ordinary solids, e.g. collecting the polymer on a filter and washing to remove by-products and unchanged products. However, the techniques described are unsatisfactory for use in a system where a colloidal suspension is employed, i.e. a latex. Reaction conditions and manipulations must be compatible for working with a colloid. The invention described herein teaches how to effect difunctional mediated couplings between a modified acrylamide latex and proteins leading to stable latex-protein products useful as diagnostic agents.